PROJECT SUMMARY Human cytomegalovirus (HCMV) is the single most important infection leading to transplant failure and continues to be a cause of substantial morbidity and death. Indeed, HCMV disease occurs in 20-30% of transplants at risk for infection and is a particular problem to lung or heart-lung recipients with a reported incidence of 50-80%. Clinical manifestations of HCMV are widespread, inflammatory in nature, and dependent on end-organ dysfunction. Inflammatory organ diseases associated with a HCMV infection is a direct consequence of the systemic viral spread to and infection of multiple organ sites that occur during either asymptomatic or symptomatic infections. Monocytes are responsible for delivering the virus into tissue and play a central role in the inflammatory state of infected organs. Since therapies against HCMV are designed to block specific steps along the virus replication cycle, the lack of HCMV replication in infected blood monocytes indicates that HCMV antiviral drugs are not effective in preventing the initial spread of the virus. In accord, prophylaxis has simply shifted the kinetics of HCMV disease to later after transplantation due to the inability of antiviral drugs to eliminate infiltrating infected inflammatory myeloid cells, which are the principle cell type found in infected organs of transplant patients. Thus, we advocate that the suppression of HCMV replication with prophylactic antiviral drugs must be administered in combination with novel drugs specifically capable of killing infected monocytes. We have found that HCMV explicitly utilizes cellular Mcl-1, an antiapoptotic member of the Bcl-2 family of proteins, to stimulate the survival of infected monocytes. Using a high- throughput screening approach, Dr. Nikolovska-Coleska?s laboratory has synthesized and characterized two novel classes of highly selective Mcl-1 antagonists. We have tested one lead compound from each class of inhibitor and demonstrated both to have high killing activity towards HCMV-infected monocytes. Furthermore, we were able to significantly enhance the selectivity and killing activity of each compound by encapsulation into nanoparticles developed in Dr. Luo?s laboratory. Thus, we hypothesize that Mcl-1 inhibitors will target HCMV- infected monocytes to prevent hematogenous dissemination, which will be tested in 3 separate aims. Aim 1 will further evaluate the two library classes of Mcl-1 small-molecule inhibitors to identify compounds exhibiting maximum cytotoxicity against HCMV-infected monocytes. Medicinal chemistry will also be preformed to increase the potency of compounds. Aim 2 will be to further develop and characterize nanoparticle technology to enhance targeting of Mcl-1 inhibitors towards infected monocytes. Aim 3 will determine the efficacy of free versus encapsulated Mcl-1 small-molecule inhibitors at neutralizing/limiting HCMV spread in a humanized mouse model. These studies will evaluate the use of Mcl-1 inhibitors as an antiviral strategy to target infected cells rather than the virus directly, which could have major impact on the prognosis of transplant patients at high-risk for HCMV infection.